Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca²⁺ and membrane ion current clocks driven by Ca²⁺-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A₁, A₃) that couple to G_i protein-coupled receptors, reducing spontaneous AP firing rate via G_βγ signaling that activates I_KAch,Ado. Adenosine also inhibits adenylyl cyclase activity via G_αi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to I_KAch,Ado activation, adenosine impacts also Ca²⁺ via G_αi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca²⁺ and membrane clock coupling. To this end, we measured Ca²⁺ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca²⁺ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca²⁺releases (LCRs) (Ca²⁺-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100 µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate G_βγ and I_KACh,Ado and to activate G_αi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.